sign in sign up
<<
Home , Frullania nisquallensis, Makinoa, Marchantiophyta, Plagiochila, Polytrichum, Polytrichum commune

Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2, 73-85 ORIGINAL ARTICLE www.jpccr.eu

Cytotoxic and Antiviral Compounds from and Inedible Fungi Yoshinori Asakawa1, Agnieszka Ludwiczuk1,2, Toshihiro Hashimoto1 1 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, Japan 2 Department of Pharmacognosy with Medicinal Laboratory, Medical University of Lublin, Poland Asakawa Y, Ludwiczuk A, Hashimoto T. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi. J Pre-Clin Clin Res. 2013; 7(2): 73–85. Abstract Over several hundred new compounds have been isolated from the bryophytes and more than 40 new carbon skeletal terpenoids and aromatic compounds found in this class. Most of liverworts elaborate characteristic odiferous, pungent and bitter tasting compounds many of which show, antimicrobial, antifungal, antiviral, allergenic contact dermatitis, cytotoxic, insecticidal, anti-HIV, superoxide anion radical release, growth regulatory, neurotrophic, NO production inhibitory, muscle relaxing, antiobesity, piscicidal and nematocidal activity. Several inedible mushrooms produce spider female pheromones, strong antioxidant, or cytotoxic compounds. The present paper concerns with the isolation of terpenoids, aromatic compounds and acetogenins from several bryophytes and inedible fungi and their cytotoxic and antiviral activity. Key words bryophytes, inedible fungi, terpenoids, bis-bibenzyls; cytotoxicity, antiviral activity

1. Chemical constituents of bryophytes

1.1 Introduction The bryophytes are found everywhere in the world except in the sea. They grow on wet soil or rock, the trunk of trees, in lake, river and even in Antarctic island. The bryophytes are placed taxonomically between algae (Fig. 1) and (Fig. 2); there are approximately 24,000 species in the world. They are further divided into three phyla, Bryophyta ( 14,000 species, Fig. 3), (liverworts 6,000 species, Fig. 4a, 4b) and Anthocerotophyta ( 300 species, Fig. 5). Although they are considered to be the oldest terrestrial plants, no strong scientific evidence for this has appeared in literatures. This hypothesis is mainly based on the resemblance of the present-day liverworts to the first land plant , the of which date back almost 500 million years. Among the bryophytes almost Figure 2.

Figure 1. Figure 3.

Address for correspondence: Yoshinori Asakawa, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, Japan all liverworts possess beautiful cellular oil bodies (Fig. 6) e-mail: [email protected] which are peculiar membrane-bound cell organelles that Received: 01 August 2013; accepted: 30 December 2013 consist of ethereal terpenoids and aromatic oils suspended 74 Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2 Yoshinori Asakawa, Agnieszka Ludwiczuk, Toshihiro Hashimoto. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

Figure 4a. Thalloid liverwort. Figure 5.

Figure 4b. Stem-leafy liverwort. Figure 6. Oil bodies of the liverwort vethii in carbohydrates- or protein-rich matrix, while the other 1.2 Bio- and Chemical Diversity of Bryophytes two phyla do not. These oil bodies are very important 1.2.1 Biodiversity of bryophytes biological marker for the in the Marchantiophyta The Marchantiophyta includes two subclasses, the [1–8]. Phytochemistry of bryophytes has been neglected for Jungermanniidae and Marchantiidae, and six orders, 49 a long time because they are morphologically very small families, 130 genera and 6,000 species. Still many new species and difficult to collect a large amount as pure sample, their have been recorded in the literatures. The southern hemisphere identification is also very difficult even under the microscope. together with tropic region is characterized by extraordinarily They are considered to be nutritionally useless to humans. high liverwort diversity [14]. This area has more endemic In fact, nothing references concerning use as foods for liverwort species than the northern hemisphere. As seen in humans have been seen. However, a number of bryophytes, Fig. 7, there are 54 endemic genera in southern hemispheric especially, mosses have been widely used as medicinal plants countries, such as New Zealand and Argentina [15]. In in China, to cure burns, bruises, external wounds, snake Asia including Japan, relatively large number of endemic bite, pulmonary tuberculosis, neurasthenia, fractures, genera (21) also has been recorded, however, South Africa, convulsions, scald, uropathy, pneumonia, neurasthenia etc. Madagascar and both North America and Europe are very as shown in Table 1 [9–11]. poor regions of endemic liverworts. The richness of endemic Many species of liverworts show characteristic fragrant odors genera of bryophytes in southern hemisphere suggests that the and an intense pungent, sweet or bitter taste. Generally, bryophytes might originate from the past Antarctic islands bryophytes are not damaged by and fungi, insect since 350 – 400 million years ago and developed to the northern larvae and adults, snails, slugs and other small mammals. hemisphere with a long range evolutionary process. In Japan, Furthermore, some liverworts cause intense allergenic Yaku Island is the most important place to watch many species contact dermatitis and allelopathy. Although liverworts of the Marchantiophyta. In the southern hemisphere, New possess such pharmacologically interesting substances, their Zealand is the most charming country to see many different isolation and the structural elucidation were neglected for species of the Marchantiophyta which are totally different almost one century. from those found in the northern Asia, including Japan. The paper concerns with the cytotoxic and antiviral activity In the tropical regions, such as south-east Asia, Borneo, of terpenoids, aromatic compounds and acetogenins from Sumatra and Papua New Guinea and Colombia, Ecuador and bryophytes [2–4, 8, 12, 13]. Venezuela, there are rain forests where so many liverworts Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2 75 Yoshinori Asakawa, Agnieszka Ludwiczuk, Toshihiro Hashimoto. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

Table 1. Medicinal bryophytes and their biological activity and effects

Musci: Biological activity and effects Bryum argenteum Antidotal, antipyretic, antirhinitic activity; for bacteriosis Cratoneuron filicinum For malum cordis (heart disease) Ditrichum pallidum For convulsions, particularly in infants Fissidens japonicum Diuretic activity; for growth of hair, burns, and choloplania (jaundice, icterus) For hemostatis, pulmonary tuberculosis, vomitus cruentus (hematemesis), bruises, and athlete’s foot dermatophytosis (dermatomycosis, Funaria hygrometrica dermomycosis) Haplocladium catillatum Antidotal, and antipyretic activity; for adenopharyngitis, pharyngitis, uropathy, mastitis, erysipelas (rose), pneumonia, urocystitis, and tympanitis Leptodictyum riparium Antipyretic; for choloplania, and uropathy Mnium cuspidatum For hematostasis and nosebleed Oreas martiana For anodyne (pain), hemostasis, external wounds, epilepsy, menorrhagia, and neurasthenia (nervosism, nervous exhaustion) Philonotis fontana Antipyretic, antidotal activity; for adenopharyngitis Plagiopus oederi As a sedative; for epilepsy, apoplexy, and cardiopathy species Diuretic activity; for hair growth Antipyretic, and antidotal; for hemostasis, cuts, bleeding from gingivae, hematemesis, and pulmonary tuberculosis Rhodobryum giganteum Antipyretic, diuretic, and antihypertensive; for sedation, neurasthenia, psychosis, cuts, cardiopathy, and expansion of heart blood vessels Rhodobryum roseum As a sedative; for neurasthenia, and cardiopathy Taxiphyllum taxirameum Antiphlogistic; for hemostasis, and external wounds Weissia viridula Antipyretic, and antidotal; for rhinitis Hepaticae: Biological activity and effects Antimicrobial, antifungal, antipyretic, antidotal activity; used to cure cuts, burns, scalds, fractures, swollen tissue, poisonous snake bites, and conicum gallstones Antiseptic activity polymorpha Antipyretic, antihepatic, antidotal, diuretic activity; used to cure cuts, fractures, poisonous snake bites, burns, scalds, and open wounds hemisphaerica For blotches, hemostasis, external wounds, and bruises

bryophytes and their structures were elucidated [2, 3, 4]. Most of compounds found in liverworts are composed of lipophilic mono-, sesqui- and diterpenoids and aromatic compounds, such as typical bibenzyls and bis-bibenzyls. The most characteristic chemical phenomenon of liverworts is that most of sesqui- and diterpenoids are enantiomers of those found in higher plants although there are a few exceptions such as germacrane- and guaiane-type sesquiterpenoids. It is very noteworthy that the different species of the same genera, like Frullania tamarisci and F. dilatata (Frullaniaceae) each produces different sesquiterpene lactone enatiomers. Some liverworts, such as species (), biosynthesize both enantiomers. Flavonoids, fatty acids and phytosterols are ubiquitous components in bryophytes and have been isolated Figure 7. The distribution of endemic liverwort genera from or detected both in liverworts and mosses. However, the presence of nitrogen or sulfur or nitrogen species have been found, but many different species like the and sulfur containing compounds in bryophytes was very species are intermingled each other and thus rare. Recently several nitrogen-sulfur containing compounds it is thus time consuming work to purify all of them. In (1–4) have been isolated from the Mediterranean liverwort, Ecuador and Columbia, the Marchantiophyta species grow in the high mountains, over 2,000m where people live, not in the lower level of their lands.

1.2.2. Chemical Diversity of Bryophytes The extraction of oil bodies with n-hexane or ether, using ultrasonic apparatus is very easy for stem-leafy liverworts to give a large amount of crude extract. In case of thalloid liverworts, the specimens are ground mechanically and then extracted with non-polar solvents. At present, over several hundred new compounds have been isolated from 76 Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2 Yoshinori Asakawa, Agnieszka Ludwiczuk, Toshihiro Hashimoto. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

Corsinia coriandrina (Corsiniaceae: ) [16], obscura, Lepidozia vitrea, subciliata, two prenyl indole derivatives (5,6) from the sciophila, semirepandus, and Trocholejeunea species () [3], skatole (7) from the or sandvicensis were inactive against this same cell line (IC50 () [3] and the Tahitian Cyathodium values >20 mg/ml) (Asakawa, unpublished results). foetidissimum (Cyathodiaceae) [8] and benzyl- (8,9) Many Plagiochila species contained cytotoxic plagiochiline and β-phenethyl β-methylthioacrylates (10) from the A (11) (0.28mg/mL) against KB cell [12]. The ether extract Isotachidaceae [3]. of Plagiochila ovalifolia showed inhibitory activity against P-388 murine leukemia cells, and its constituents, plagiochiline A (11), plagiochiline A-15-yl octanoate (12), and 14-hydroxyplagiochiline A-15-yl (2E,4E)-dodecadienoate (13) m exhibited IC50 values of 3.0, 0.05, and 0.05 g/mL, respectively [17]. Compound 12 and plagiochiline A-15-yl decanoate (14) from P. ovalifolia, polygodial (15) from P. vernicosa complex, as well as sacculatal (16) from P. endiviifolia showed

cytotoxic activity against a human melanoma cell line (IC50 Highly evolved liverworts belonging to the value range 2–4 mg/mL). Compound 16 was cytotoxic also m produce phytosterols, such as campe-, for Lu1 (IC50 5.7 g/mL), KB (3.2), LNCaP (7.6), and ZR- stigma- and sitosterols. Almost all liverworts elaborate 75–1 cells (7.6) (Cordell, Pezzuto, Asakawa, unpublished a-tocopherol and squalene. The characteristic components results). Lepidozenolide (17) showed potent cytotoxicity of the Bryophyta are highly unsaturated fatty acids when evaluated in the P-388 murine leukemia cell line (IC50 and alkanones, such as 5,8,11,14,17-eicosapentaenoic 2.1 mg/mL) [18]. acid, 7,10,13,16,19-docosapentaenoic acid and 2α,5β-Dihydroxybornane-2-cinnamate (18) from 10,13,16-nonadecatrien-7-yn-2-one and triterpenoids. The and lunularin (19) from Dumortiera neolignan is one of the most important chemical markers hirsuta, exhibited cytotoxic activity against human HepG2 m of the Anthocerotophyta [3]. cells, with IC50 values of 4.5 and 7.4 g/mL respectively [19]. The presence of hydrophobic terpenoids is very rare in the hirtellus produces a new sesquiterpene Marchantiophyta. A few bitter kaurene glycosides have been lactone, chandolide (20) [20], 13,18,20-tri-epi- found in the species. However a numbers of chandonanthone (21) [21] and anadensin (22) which were flavonoid glycosides have been detected both in liverwort evaluated for cytotoxic activity against the HL-60 leukemia and mosses [2, 3, 4]. cell line, and exhibited IC50 values, in turn, of 5.3, 18.1, and 17.0 mg/mL. 6α-Methoxyfusicoauritone (23) isolated from 1.3. Bioactive compounds from bryophytes the same liverwort showed some cytotoxicity against KB m The biological characteristics of the terpenoids and aromatic cells (IC50 11.2 g/mL), although compounds 21 and 22 were compounds isolated from the liverworts in our laboratory inactive [20,22]. are: 1) characteristic scents, 2) pungency and bitterness, 13-Hydroxychiloscyphone (24) from Chilosyphus rivularis, 3) allergenic contact dermatitis, 4) cytotoxic, 5) antimicrobial, was tested against the RS322, RS188N, and RS321 yeast m antifungal and antiviral, 6) insect antifeedant, mortality, strains. It showed IC12 values of 75 and 88 g/mL for strains and nematocidal, 7) superoxide anion radical release RS321 and RS322. These data are characteristic of a selective inhibitory, 8) 5-lipoxygenase, calmodulin, hyaluronidase, DNA-damaging agent that does not act as a topoisomerase cyclooxygenase, DNA polymerase b and a-glucosidase I or II inhibitor. Compound 24 also showed cytotoxic inhibitory, 9) antioxidant 10) piscicidal 11) neurotrophic, 12) muscle relaxing and calcium inhibitory, 13) cardiotonic and vasopressin antagonist, 14) liver X-receptor (LXR)a agonist and LXRb antagonist, 15) cathepsins B and L inhibitory, antithrombin, 16) farnesoid X-receptor (FXR) activation, 17) nitric oxide production inhibitory, 18) plant growth inhibitory, 19) tublin polymerization inhibitory, 20) sex pheromones and 21) antiplatelet and brine shrimp lethal activity.

1.3.1 Cytotoxic compounds from bryophytes Several sesquiterpene lactones, such as eudesmanolides, germacranolides, and guaianolides isolated from liverworts exhibit cytotoxic activity against KB nasopharyngeal and P-388 lymphocytic leukemia cells (Asakawa 1995). The crude ether extracts of the liverworts pompeana, makinoana, heterophylla, crispata, emarginata, endiviifolia, Plagiochila fruticosa, P. ovalifolia, caespitans, P. japonica, P. perrottetiana, P. vernicosa, and perrottetii showed cytotoxicity against P-388 cells (IC50 value range 4–20 mg/mL). In contrast, the crude extracts of Frullania diversitexta, F. ericoides, F. muscicola, F. tamarisci subsp. Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2 77 Yoshinori Asakawa, Agnieszka Ludwiczuk, Toshihiro Hashimoto. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

against both HL 60 and KB cells. However, (-)-diplophyllin (38) did not indicate cytotoxicity against either of these cell lines [28]. Glaucescenolide (39) from glaucescens showed cytotoxic activity against P-388 mouse leukemia cells µ β (IC50 2.3 g/mL) [29]. ent-1 -Hydroxykauran-12-one (40), isolated from Paraschistochila pinnatifolia and 1α-hydroxy- ent-sandaracopimara-8(14),15-diene (41) from m mollissima, showed IC50 values of 15 and >25 g/mL, when evaluated against this same cell line [30]. The ethanol-soluble extract of Lepidolaena taylorii which m showed cytotoxicity against P-388 cell line, (IC50 1.3 g/mL), was purified to give the 8,9-secokaurane diterpenoids, activity against lung carcinoma A-549 cells (IC50 value rabdoumbrosanin (43), 16,17-dihydrorabdoumbrosanin 2.0 mg/mL) [23]. (43), 8,14-epoxyrabdoumbrosanin (44) and their related (-)-ent-Arbusculin B (25) and (-)-ent-costunolide (26) from compounds 45–48 and the ent-kaur-16-en-15-ones (49–53). Hepatostolonophora paucistipula, showed cytotoxic activity In turn, L. palpebrifolia also elaborated the 8,9-secokauranes against P388 murine leukemia cells, with IC50 values of 1.1 (42, 44). The cytotoxicity of these ent-8,9-seco and ent- and 0.7 mg/mL [24]. Costunolide (27) isolated from Frullania kaurenes was tested against the mouse P-388 leukemia and nisqualensis showed growth inhibitory activity against the several human tumor cell lines, inclusive of six leukemia and

A-549 human lung carcinoma cell line with an IC50 value a range of organ-specific cancer cell lines. Compounds 42 of 12 mg/mL and moderate, but selective, DNA-damaging and 44 showed the most potent cytotoxic activities (mean m activity against the RS321N, RS322YK, and RS167K mutant IC50 values of 0.006 and 0.27 g/mL; GI50 values of 0.10 and m m yeast strains, with IC12 values of 50, 150, and 330 g/mL 1.2 M, respectively). Compound 44 also showed cytotoxicity [25]. Naviculyl caffeate 28( ), from Bazzania novae-zelandiae, against P-388 cell at 0.80 mM.). Compounds 42 (including demonstrated growth inhibitory effects against P-388 murine 10% of 43) and 44 showed differential cytotoxicity in vitro m leukemia cells with a GI50 value of 0.8–1.1 g/mL, although when tested against five further leukemia cell lines with 41 m naviculol (29) was inactive [26]. Riccardiphenol C (30), showing an average IC50 value of 0.4 M; however, cell growth m from Riccardia crassa showed slight cytotoxicity against was not inhibited by 44 (IC50 >50 M). The growth of seven BSC-1 (African green monkey kidney epithelial) cells at colon cancer cell lines was inhibited also by 42 (mean IC50 60 mg/disk [27]. value, 6 mM) [31,32]. Compounds 42 and 44 were tested The ether and methanol extracts of the Tahitian in an in vivo hollow fiber model system, in which neither Mastigophora diclados showed cytotoxic activity against compound was active at the doses tested (18 and 12 mg/kg for m m HL 60 cells at IC50 2.4 and 13.1 g/mL and KB cells at 42 and 150 and 100 g/kg for 44). Compound 48 was most m m 14.6 and 32.5 g/mL, respectively [28]. (-)-Diplophyllolide active against several leukemia cell lines (mean GI50 0.3 M) (31), a-herbertenol (32), (-)-herbertene-1,2-diol (33), and least active against various central nervous system cancer m mastigophorene C (34) and mastigophorene D (35) isolated cell lines (mean GI50 6 M) [32]. from both extracts were cytotoxic against HL 60 cells with Ent-kaurene (54) and (55) from the New Zealand m IC50 values of 2.5, 1.4, 12.8, 1.4, and 2.4 g/mL. They also Jungermannia species showed weak cytotoxic activity against m showed cytotoxicity against KB cells (IC50 values of 14.2, 3.3, P-388 at 0.48 and 25 g/mL, respectively [33] Among the 12.5, 11.8 and 14.8 mg/mL). 2-Methoxy (36) and diacetoxy isolated compounds, 8,9-secokaurenes (42, 44, 48) showed derivatives (37) of herbertane-1,2-diol (33) showed evidence selective toxicity amongst human tumor cell lines at a of having less potent cytotoxicity than the parent compound concentration of 1.2, 2.5 and 1.5 mM, respectively. 78 Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2 Yoshinori Asakawa, Agnieszka Ludwiczuk, Toshihiro Hashimoto. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

The mode of action for the cytotoxicity of the ent-8,9- 61 possessed the most potent cytotoxic activity against HL- m secokaur-16-en-15-one and ent-kaur-16-en-15-one series was 60 and KB cells showing IC50 values of 0.92 and 0.96 M. supported by Michael addition of a thiol to the C-16–C-17 The other compounds 63–65( ) and the 6’-nitro derivative double bond of 42, but the C-8–C-14 double bond of 43 was of 63 indicated much less activity against both cell lines m relatively unreactive [31,32]. (HL-60 IC50 value range, 6.3–96.6 M; KB IC50 value range, Clavigerins A-D (56–59) isolated from Lepidolaena 5.5–124.3 mM). From the Tahitian sample, costunolide clavigera showed a weak cytotoxicity (30mg/disk) against (27) and tulipinolide (67) were obtained and the former BSC cells [34]. germacranolide shown to be cytotoxic against the HL-60 m A new atisane-2 derivative (60) from Lepidolaena clavigera cell line (IC50 4.6 M) [38]. exhibited weak inhibitory activity against mouse lymphocytic Porella perrottetiana produced cytotoxic compounds m leukemia cells (P-388) with an IC50 value of 16 g/mL [35] against both HL-60 and KB cell lines [38]. The same [35]. α-Zeorin (61) has been isolated from several liverworts treatment as mentioned above gave 4α,5β-epoxy-8-epi- and displayed cytotoxic activity against P-388 cells with an inunolide (68), 7-keto-8-carbomethoxypinguisenol (69) and m IC50 of 1.1 g/ml [36,37]. perrottetianal A (70). The former two compounds exhibited moderate or weak cytotoxicity against HL-60 (IC50 8.5 and m m α 2.7 M) and KB cells (IC50 52.4 and 46.3 M). 7 -Hydroxy- 8-carbomethoxypinguisenol (71a) and acutifolone A (71b) prepared from 69 by reduction and dehydration were m evaluated against HL-60 (IC50 83.10 and >177 M) and KB m cells (IC50 2.7 and 46.6 M). It was suggested that the dienone group plays an important role in the mediation of cytotoxcity against HL-60 cells [38].

The crude ether extract of two unidentified Indonesian and Tahitian Frullania species exhibited cytotoxic activity against both the HL-60 and KB cell lines, with at EC50 values of 6.7 and 1.6 mg/mL (HL-60 cells) and 1.6 and 11.2 mg/mL (KB cells), respectively [38]. Bioactivity-guided fractionation of the Indonesian sample led to the isolation of (+)-3α-(4’-methoxybenzyl)-5,7-dimethoxyphthalide (62), (-)-3α-(3’-methoxy-4’,5’-methylenedioxybenzyl)-5,7- dimethoxyphthalide (63), together with 3-methoxy-3’,4’- Macrocyclic bis-bibenzyls such as marchantin A (72) and methylenedioxybibenzyl (64), 2,3,5-trimethoxy-9,10- riccardin A (73) were firstly isolated from liverworts by dihydrophenanthrene (65) and atranorin (66), among which Asakawa et al.[1,2]. Up to now, more than 60 macrocyclic and acyclic bis-bibenzyls have been isolated from many liverworts and their stereostructures established [3,12,39]. The cyclic bis-bibenzyls such as marchantin (e.g.72) and riccardin series (e.g.73) might be biosynthesized from bibenzyls that correspond chemically to dihydrostilbenes [40]. This assumption was proved by feeding experiments using radioactive and 13C-labelled precursors, like L-[U- 14C]-phenylalanine, [U-14C]dihydro-p-coumaric acid, [2– 13C]acetate, and L-[13COOH]phenylalanine. Marchantin C (74) was biosynthesized by coupling of two lunularic acid (80), followed by cytochrome P-450, named marchantin C hydroxylase to afford marchantin A 72( ) [4]. Macrocyclic bis-bibenzyls biosynthesized by liverworts possess various biological activities, like antimicrobial, antifungal, muscle relaxant, cytotoxicity against KB cells, inhibitory activity against DNA-polymerase b, cardiovascular activity, anti-HIV, and antitumor activity [2,3, 13] The methanol extract (105 g) of a Japanese specimen of M. polymorpha was chromatographed over silica gel and Sephadex LH-20 to give the cyclic bis-bibenzyls, marchantin A (72) (30 g), and its analogues, marchantins B (75), C (74), D (76), E (77), G (78), J (79). The yield of 72 is dependent Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2 79 Yoshinori Asakawa, Agnieszka Ludwiczuk, Toshihiro Hashimoto. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

upon the particular Marchantia species being investigated. Pure 72 (80 to 120 g) has been isolated from 6.67 kg of dried M. paleacea var. diptera[4]. This thalloid liverwort elaborates not only the marchantin series, including marchantins A (72), B (75), D (76), and E (77), but also the acyclic bis-bibenzyls, perrottetin F (81) and paleatin B (82). Marchantins A (72), B (75), D (76), perrottetin F (81), and paleatin B (82) showed cytotoxicity against KB m cells (IC50 range 3.7–20 M) and P-388 (T/C 117) [13].

effects in reversing P-glycoprotein-mediated multidrug resistance [44]. 2-Hydroxy-3,4,6-trimethoxyacetophenone (88) and 2-hydroxy-4,6-dimethoxyacetophenone (89) from Plagiochila

fasciculata, were inactive against the P-388 cell line (IC50 Marchantin A (72) induced cell growth inhibition in human values of >50 mg/mL) [46]. m MCF-7 breast cancer cells at IC50 4.0 g/mL. Fluorescence Trichocolea lanata and T. tomentella produced tomentellin microscopic and a Western blot analysis indicated that (90), which showed inhibitory activity against African green compound 72 induced apoptosis of MCF-7 cells through a monkey kidney epithelial (BSC-1) cells at 15 mg/mL, with caspase-dependent pathway. The phenolic hydroxy groups no antiviral effects against herpes simplex or polio viruses. at C-1' and C-6' are responsible for inducing cytotoxic and Demethoxytomentellin (91) from T. tomentella showed a antioxidant activity [41]. similar cell growth inhibitory effect, indicating that both Marchantin C (74) and its dimethyl ether, 7,8-dehydro- an allylic ether and a conjugated enone substructure are marchantin C and its dimethyl ether were synthesized required for such activity [47]. Methyl-4-[(2E)-3,7-dimethyl- and their possible modulatory effects on P-glycoprotein in VCR-resistant KB/VCR cells were investigated [42]. The results indicated that 74 was the most potent inhibitor of cell proliferation in both KB and KB/VCR cells among these four synthetic compounds, while the three derivatives of 74 has little antiproliferative activity. Potent apoptosis in KB/VCR cells was induced by treatment with 16 µM of dimethyl ether of marchantin C (74) and 0.2 µM VCR for 48 hours [42]. Marchantin C also showed the induction of apoptosis of human glioma A172 cells at 8–16mM [43]. Marchantin C (74), neomarchantins A (83) and B (84), and a mixture of sesquiterpene/bis-bibenzyl dimers, GBB A (85) and GBB B (86) from Schistochila glaucescens showed growth inhibitory activity against the P-388 cell line, with IC50 values of 18, 7.6, 8.5, and 10.3 mg/mL, respectively [29]. Riccardin D (87) from Monocolea forsteri [3] and [44, 45] indicated antiproliferative activity on human glioma A172 cells and induction of apoptosis at 16 mM. Compound 87 also possesses potent 80 Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2 Yoshinori Asakawa, Agnieszka Ludwiczuk, Toshihiro Hashimoto. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

2,6-octadienyl]oxy]3-hydroxybenzoate (92), isolated from Theent -kaurenes 54, 101–106, isolated from the Japanese m T. hatcheri, showed a lack of cytotoxicity (IC50 >100 M) liverwort Jungermannia truncata, were evaluated for against both KB and SK-MEL-3 human melanoma cells, as cytotoxicity against HL-60 human leukemia cells. Of these, well as NIT 3T3 fibroblasts [48] ent-11a-hydroxy-16-kauren-15-one (54) induced apoptosis (programmed cell death) in this cell line partly through a caspase-8 dependent pathway [33,51]. The presence of an enone group in this class of molecule appears to be essential for the induction of apoptosis and the activation of caspases in human leukemia cell lines [49,52]. ent-Kaurenes 54, 50 and 105 and ent-9(11),16-kauradien- 12,15-dione (93), and the rearranged ent-kaurene, jungermannenone A (95), selectively inhibited nuclear factor- mB (NF-mB)-dependent gene expression due to treatment with TNF-α. Compound 54, in combination with TNF-α caused a dramatic increase in apoptosis in human leukemia cells accompanied by activation of caspases. Compound 54, when combined with camptothecin, also caused an increase in apoptosis [53,54]. Jungermanenones A-D (95–100), obtained from Jungermannia species, induced cytotoxicity against human leukemia HL-60 cells at 50% inhibitory concentrations of 1.3, 5.5, 7.8, and 2.7 mM, respectively, and DNA fragmentation and nuclear condensation. Both are biochemical markers of apoptosis induction, and apoptosis was induced through a caspase-independent pathway. Compounds 95 and 100 showed inhibitory activity for NF-kB, which is a transcriptional factor of antiapoptotic factors. Thus, ent-kaurene diterpenoids from liverworts may Theent -kauranes and kaurenes (54, 93–95) isolated from be promising candidates as antitumor agents [55,56].

Jungermannia species inhibited HL-60 cells with IC50 values, Some monoterpenoids, such as bornyl acetate (107) in turn, of 0.49, 7.0, 0.59, and 0.28 mM. Treatment of 54 and found in liverworts demonstrate potent apoptosis- 93–95 caused proteolysis of poly(ADP-ribose) polymerase, inducing activities against the cultured cells of Marchantia a sign of activation of the apoptotic machinery, whereas the polymorpha. Apoptosis induced by monoterpenoids occurs feature of cell death induced by treatment with compounds 93 via the production of active oxygen species such as H2O2 [57]. and 94 was necrosis. Treatment with compound 95 induced The ursane triterpenoids from the liverwort apoptosis (see below) [49]. The ent-kaurane diterpenoids pulcherrimum, ursolic acid (108), acetoxyursolic acid (109), 96, 97 and 98–100 from a Jungermannia species showed and 2α,3β-dihydroxyurs-12-en-28-oic acid (110) showed cytotoxicity for HL-60 cells with IC50 values of 1.00, 0.40, inhibition of the growth of PC3 human prostate cancer cells, 1.21, 1.28, and 0.78 µM, respectively [50]. at concentrations between 10.1±1.00 and 39.7±2.98 mM [58]. Previously, the two pimarane diterpenoids momilactones A (111) and B (112), which were identified as phytoalexins in rice, have been isolated from the moss Hypnum plumaeforme (Hypnaceae) [59]. Momilactone B (112) was shown to have cytotoxicity against human colon cancer HT-29 and SW620 cells at 1mM [60]. Pallidisetin A (113) and pallidisetin B (114), isolated from the moss Polytrichum pallidiscetum, showed cytotoxicity against human melanoma (RPMI-7951) and human Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2 81 Yoshinori Asakawa, Agnieszka Ludwiczuk, Toshihiro Hashimoto. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

were evaluated using a PA endonuclease inhibition assay in vitro [64]. Among them, the bis-bibenzyls, marchantins A (72), B (75), and E (77) and plagiochin A (123) inhibited influenza PA endonuclease activity at a concentration of 10 mM. This was the first evidence that the phytochemicals derived from liverworts can inhibit influenza A endonuclease. pusilla produces the bis-bibenzyl dimers, pusilatins A-D (124–127). Pusilatins B (125) and C (126) were found

to possess DNA polymerase b inhibitory activity (IC50 values of 13.0 and 5.16 mM, respectively) and showed weak HIV-RT inhibitory activity [65] as shown in Table 2.

Table 2. Cytotoxic and anti-viral bis-bibenzyls from bryophytes against KB cells, DNA polymerase b and HIV-1 glioblastoma multiforme (U-251 MG) cells, with ED50 values of 1.0 and 1.0 mg/mL and 2.0 and 2.0 mg/mL [61]. Bis-bibenzyls KB KB+VLB* KB-VLB* DNA polymerase-b HIV-1 Three cytotoxic compounds, 1-O-methylohioensin B (mM) (mM) (mM) (mM) (mg/mL) (115), 1-O-methyldihydroohioensin B (116) and 1,14-di- Marchantin A (72) 3.7 1.3 2.7 97.5 11.5 O-methyldihydroohioensin B (118) were also isolated from Marchantin B (75) 3.2 9.3 4.0 14.4 9.3 the moss Polytrichum pallidisetum. Compound 115 proved Marchantin D (76) 10.8 >20.0 >20.0 55.4 23.7 to be cytotoxic for human colon adenocarcinoma (HT-29), Marchantin E (77) 7.6 1.6 2.9 20.0 21.2 human melanoma (RPMI-7951), and human glioblastoma Perrottetin F (81) >20.0 12.6 9.5 14.3 5.3 multiforme (U-251 MG) cells, with ED50 values of 1.0, 1.0, and 2.0 mg/mL. Compound 116 showed inhibitory activity only Paleatin B (82) >20.0 >20.0 >20.0 18.5 22.1 Pusilatin B (125) 13.1 15.3 11.9 13.0 ** against U-251 cells (ED50 0.8 mg/mL) while 118 inhibited the m Pusilatin C (126) 13.8 7.1 11.7 5.16 ** growth of the A549 lung carcinoma (A549) (ED50 1.0 g/mL) m and RPMI-7951 melanoma (ED50 1.0 g/mL) cell lines [61]. * VLB: Vinblastine: ** not tested Ohioensin H (117) from Polytrichum commune did not show any cytotoxicity against the five human cancer cell lines in m which it was evaluated (IC50 in all cases >5 g/mL) [62]. 1.3.3 Tubulin polymerization inhibition Marsupellone (119) and acetoxymarsupellone (120) Marchantin C (74) strongly inhibited the growth of human from Marsupella emarginata showed cytotoxicity (ID50 cervical tumor xenografts in nude mouse and decreased the 1mg/mL) against P388 [10]. Riccardins A (73) and B (121) quantity of microtubules in a time- and dose-dependent which were the first bis-bibenzyls from the Japanese manner at the G2/M phase in human glioma tumor cells liverwort, subsp. decrescens inhibited and HeLa (human cervical adenocarcinoma cell line) cells KB cells at a concentration of 10 and 12 mg/mL, respectively. at 8–16 mM [43, 66]. The same compound 74( ) decreased contained cytotoxic perrottetin E (122) the polymerization rate of gross tubulin, similar to the (12.5 mg/mL) against KB cells [10]. microtubule depolymerizor, vincristine, at 8–24 mM. These results indicated that 74 plays the same role in microtubule 1.3.2 Antiviral compounds from bryophytes depolymerization in both its apoptotic effects in the cell Marchantins A (72), B (75), D (76), perrottetin F (81), and and subsequent antitumor activity in vivo. Compound paleatin B (82) showed anti-HIV-1 activity (IC50 range 5.3– 74 is a novel microtubule inhibitor that induces mitotic 23.7 mg/mL) [10,63] as shown in Table 2. arrest of tumor cells and suppresses tumor cell growth. The H1N1 and H5N1 influenza A virus caused pandemics The structure of marchantin C is distinct from classical throughout the world in 2009. Influenza A possesses an microtubule inhibitors like colchicine, paclitaxel, vinblastine, endonuclease within its RNA polymerase comprised of PA, and vincristine. However, this macrocyclic bis-bibenzyl may PB1, and PB2 subunits. In to obtain potential new anti- be regarded as a potential antitumor agent as a result of influenza compounds, 33 different types of phytochemicals inhibiting microtubule polymerization [43,66]. 82 Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2 Yoshinori Asakawa, Agnieszka Ludwiczuk, Toshihiro Hashimoto. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

Isoplagiochins A (128) and B (129) isolated from Plagiochila fruticosa inhibited the polymerization of tubulin with IC50 values of 50 and 25 mM. The dihydro derivatives of both m 128 and 129 were found to be inactive (IC50 >100 M), and, when compared with the parent compounds, indicated that a restricted biaryl ring system is favorable for tubulin binding. A Monte Carlo search showed that the presence of two aromatic rings connected by a two-carbon bridge with a double bond may serve to maintain the backbone conformation [67].

Figure 8. Cryptoporus volvatus (Polyporaceae) on dead pine tree.

- leucocyte induced by O2 radical stimulant FMLP at a 2. Chemical constituents of inedible fungi concentration of 6 mg/mL [70]. Those bitter drimanes with isocitric acid moiety showed cytotoxicity against KB, Lui, 2.1 Introduction LNCaP ad ZR-75–1 cancer cell lines as shown in Table 3. There are about 1500 identified fungi in Japan among which The cytotoxicity of the permethylated CPA series is more 300 species are edible, 1150 inedible and 50 toxic. The chemical potent than that of their naturally occurring CPAs [Asakawa constituents of toxic and edible fungi have been fully studied. & Hahimoto, unpublished results]. Recently, many biologically interesting compounds were The same CPA series 130–136( ) showed inhibitory isolated from inedible mushrooms by our group and their effect at a concentration of >50, >50, 40, >50, >50, >50 structures and biological activity reported [68,69]. and >50 mg/mL against lysosome enzyme release from rat peritoneal neutrophil cell stimulated by FMLP (10–6 M) and 2.2 Bioactive compounds from a few inedible fungi cytochalasin B (5 mg/ml) [70]. The biological properties of the terpenoids and aromatic compounds and acetogenins isolated from inedible fungi in our laboratory are: 1) cytotoxic, 2) anti-HIV-1, 3) anti-HSV, 4) antimicrobial and antifungal, 5) antitumor promotion, 6) insecticidal, 7) nematocidal, 8) antifeedant against snail and slug, 9) plant growth inhibitory, 10) anti-Alzheimer disease, 11) superoxide anion release inhibitory, 12) anticholestemic activity, and 13) spider female sex pheromone production.

2.2.1 Cytotoxic compounds from Cryptoporus volvatus and Daldinia species The Cryptoporus volvatus belonging to the Polyporaceae grows on decayed pine tree and its bruiting body emits resinous smell by which insects such as Parabolitophagus felix and Ischnodactylus loripes are attracted. The wet and dried fruiting bodies contain surprisingly strong bitter principles. The fractionation of the ethyl acetate extracts from 40kg of wet fungus gave 20g order of cryptoporic acids A (=CPA-A) (130), CPA-B (131), CPA-C (132), CPA-D (133), CPA-F (135), CPA-G (136), together with CPA-E (134, 700g) as the major component. Compounds 130–136 indicated CPA-F (135), CPA-G (136). Compounds 130–136 indicated very strong inhibition effect for superoxide anion radical from guinea- - pig macrophage induced by O2 radical stimulant formyl methionyl leucyl phenylalanine (FMLP) at a concentration of 13, 25, 0.07, 0.1, 0.05, 0.3 and 0.15 mg/mL, respectively. CPA-C (133) also possessed the 88% of inhibitory effect for superoxide anion release from rabbit polymorphonuclear Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2 83 Yoshinori Asakawa, Agnieszka Ludwiczuk, Toshihiro Hashimoto. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

Table 6. Anti-HIV I activity of daurichomenic acid and grifolins Table 3. Cytotoxicity testing (ED50: mg/ml) of cryptoporic acids A-G and their methylated derivatives on KB, KB-V, Lu1, LNCaP and ZR-75–1 cell Compounds ED mg/mLa CC mg/mLb lines. 50 50 Grifolic acid (142) 40 40 a b Samples KB KB-V (+VLB) KB-V (-VLB) Lu1 LNCaP ZR-75-1 Grifolic acid methyl ether(143) 53 53 Cryptoporic acid-A (130) >20 >20 >20 >20 >20 >20 Grifolin (144) 38 38 CPA-A Me3 2.3 2.6 3.4 >20 >20 12.2 (-)-Daurichromenic acid (145) 0.00056 557 CPA-B(131) >20 >20 >20 >20 >20 >20 CPA-B Me3 >20 3.8 >20 >20 >20 >20 a: 50% effective concentration; b: 50% cytotoxic concentration CPA-C(132) >20 >20 >20 >20 >20 12.9 CPA-C Me5 14.2 3.8 >20 >20 15.7 13.0 CPA-D(133) >20 >20 >20 >20 >20 14.5 CPA-D Me4 >20 >20 >20 >20 >20 >20 CPA-E(134) >20 >20 >20 >20 >20 >20 CPA-E Me5 >20 >20 >20 >20 >20 >20 CPA-F(135) >20 >20 >20 >20 >20 >20 CPA-G(136) >20 >20 >20 >20 >20 >20 a: +VLB = presence of vinblastine; b: -VLB = absence of vinblastine

An unidentified Daldinia species belonging to the Xylariaceae produces cytochalasin series belonging to 10-pheyl-(11) class of cytochalasins and 10-phenyl-22-oxa- (12)-cytochalasin [71]. Compounds 137–141 exhibited the cytotoxic activity against KB cell line as shown in Table 4 [Hashimto & Asakawa, unpublished results].

Table 4. Cytotoxic effect of cytochalasins against KB cells

Cytochalasins Inhibition % KB cell at 10–5M KB cell at 10–6M 137 96 58 138 84 50 139 89 44 140 84 50 141 96 28

2.2.2 Antiviral compounds from inedible fungi The cryptoporic acids isolated from C. volvatus showed not only cytotoxicity against cancer cell lines but also inhibitory activity against HIV 1 RT (p66) as shown in Table 5. Among which, two permethylated products, cryptoporic acid B (131) trimethyl ether and cryptoporic acid E (134) pentamethyl (144). Acidic treatment of 142 gave racemate daurichromenic ether showed highest HIV-1 inhibitory activity [Hashimoto acids which was purified by HPLC using chiral column & Asakawa, unpublished results]. to give natural (-)-daurichromenic acid (145) [Hashimoto & Asakawa, unpublished results]. Lycogala epidendrum, Table 5. Inhibitory effect of cryptoporic acids A-G and their derivatives a smile mould belonging to the Myxomycetes elaborates on HIV-1 RT (p66). unique pyrroledicarboxylates attached to two indoles,,

2 Samples % Inhibitory at IC50 (mg/mL), r named lycogarubins A-C (146–148) of which compound 20 mg/mL 148 showed anti-HSV virus activity at a concentration of m Cryptoporic acid A (=CPA-A) (130) 16.10 Inactive IC50 17.2 g/mL in vitro [68]. CPA-A Me3 2.23 Inactive CPA-B(131) 5.77 Inactive CPA-B Me3 2.23 61.0 mg/mL, r2=0.992 CPA-C (132) 99.6 Inactive CPA-C Me5 0.00 Inactive CPA-D (133) 25.40 Inactive CPA-D Me4 0.00 Inactive CPA-E (134) 19.00 Inactive CPA-E Me5 3.75 42.2 mg/mL, r2=0.889 CPA-F (135) 99.7 Inactive CPA-G (136) 37.3 Inactive

Kashiwada et al. [72] reported that Rododendron dauriaum contained (-)-daurichromenic aicd (145) which showed potent anti-HIV activity as shown in Table 6. The inedible fungous Albatrellus dispansus produces a large amount of grifolic acid (146), along with grifolic acid methyl ether (143) and grifolin 84 Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2 Yoshinori Asakawa, Agnieszka Ludwiczuk, Toshihiro Hashimoto. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

3. Conclusion 14. Mohamed H, Baki BB, Nasrulhaq-Boyce A, Lee PK Y. eds. In: in the new millenium. Kuala Lumpur, University of Malaya, 2008.p.1–513. 15. Inoue H. Bryophytes as a indicator of continental drift (Gondwana The bryophytes and inedible fungi are found in whole the land). Kagakuasahi 1988; 8: 116–121. world. They are generally very tiny organisms. However, they 16. von Reuβ SH, König WA. Corsifurans A-C, 2-arylbenzofurans of produce a great number of secondary metabolites, including presumed stilbenoid origin from Corsinia coriandrina (Hepaticae). pungent and bitter terpenoids and polyphenolic compounds Phytochemistry 2004; 65: 3113–3118. 17. Toyota M, Tanimura K, Asakawa Y. Cytotoxic 2,3-Secoaromadendrane- or nitrogen containing compounds many of which show type sesquiterpenoids from the liverwort Plagiochila ovalifolia. Planta interesting biological activity such as cytotoxicity, antiviral Med. 1998; 64: 462–464. activity. Only 5% of the total bryophytes have been chemically 18. Shu YF, Wei HC, Wu CL. Sesquiterpenoids from liverworts Lepidozia investigated since 20 century. Further search on the secondary vitrea and L. fauriana. Phytochemistry 1994; 37: 773–776. metabolites of bryophytes and inedible fungi will results in 19. Lu, Z. -Q., Fan, P. -H., Ji, M., Lou, H. -X. Terpenoids and bisbibenzyls from Chinese liverworts Conocephalum conicum and Dumortiera the discovery of many different compounds with biologically hirsuta. J. Asian Nat. Prod. Res. 2006; 8: 187–192. and pharmaceutically interest. 20. Komala I. Ito T, Nagashima F, Yagi Y, Asakawa Y, Cytotoxic, radical scavenging and antimicrobial activities of sesquiterpenoids Acknowledgements from the Tahitian liverwort Mastigophora diclados (Brid.) Nees (Mastigophoraceae). J Nat Med. 2010; 64: 417–422. The author thank the Emeritus Prof. G. A. Cordell (Illinois 21. Wang Y, Harrison LJ, Tan BC. Terpenoids from the liverwort Univ. at Chicago), Prof. J. M. Pezzuto (Univ. of Hawaii, Chandonanthus hirtellus. Tetrahedron 2009; 65: 4035–4043. Hilo), and Prof. T. Kuzuhara for their biological test of 22. Komala I, Ito T, Nagashima F, Yagi Y, Asakawa Y. New sesqui- and bis-bibenzyls. Thanks are indebted to Prof. R. Gradstein diterpenoids from the liverwort Chandonanthus hirtellus. The 53rd (National Museum of Natural History, Paris), Dr. M, Symposium on Chemistry of Terpenes, Essential Oils and Aromatics; November 7–9, 2009; Nara, Japan. Symposium Papers, p.266–268. Mizutani (Hattori Botanical Laboratory, Nichinan, Japan) 23. Wu C, Gunatilaka AAL, McCabe FL, Johnson RK, Spjut RW, Kingston for their collection and identification of liverworts. A part of DG.I. Bioactive and other sesquiterpenes from rivularis. this work was supported by a Grant-in-Aid for the Scientific J Nat Prod. 1997; 60: 1281–1286. Research (A) (No. 11309012), and the Open and Senryaku 24. Baek SH, Perry NB, Lorimer SD, ent-Costunolide from the liverwort Hepatostolonophora paucistipula. J. Chem. Research (S). 2003; 14–15. Research from the Ministry of Education, Culture, Sports, 25. Kim, Y. C., da S. Bolzani, V., Baj, N., Gunatilaka, A. A. L., Kingston, Science and Technology. D. G. I. A DNA-damaging sesquiterpene and other constituents from . Planta Med. 1996; 62: 61–63. 26. Burgess EJ, Larsen L, Perry NB. A Cytotoxic sesquiterpene caffeate from References the liverwort Bazzania novae-zelandiae. J Nat Prod. 2000; 63: 537–539. 27. Perry NB, Foster LM. Sesquiterpene/quinol from a New Zealand 1. Asakawa Y, Huneck S, Toyota M, Takemoto T, Suire C. Mono- and liverwort, Riccardia crassa. J Nat Prod. 1995; 58: 1131–1135. sesquiterpenes from Porella arboris-vitae. J Hatt Bot Lab. 1979; 46: 28. Komala I, Ito T, Nagashima F, Yagi Y, Kawahata M, Yamaguchi K, et al. 163–167. Zierane sesquiterpene lactone, cembrane and fusicoccane diterpenoids, 2. Asakawa Y. Chemical constituents of the Hepaticae. In: Herz W, from the Tahitian liverwort Chandonanthus hirtellus. Phytochemistry Grisebach H, Kirby G.W, eds. Progress in the chemistry of organic 2010; 71: 1387–1394. natural products. Vienna, Springer, 1982. 42, p.1–285. 29. Scher JM, Burgess EJ, Lorimer SD, Perry NB. A cytotoxic sesquiterpene 3. Asakawa Y. Chemical constituents of the bryophytes. In: Herz W, Kirby and unprecedented sesquiterpene-bisbibenzyl compounds from the WB, Moore RE, Steglich W, Tamm Ch. eds. Progress in the chemistry liverwort Schistochila glaucescens. Tetrahedron 2002; 58: 7875–7882. of organic natural products. Vienna, Springer, 1995.p.1- 618. 30. Lorimer, S. D., Perry, N. B., Burgess, E. J., Foster, L. M. 1-Hydroxyditerpenes 4. Asakawa Y, Ludwiczuk A, Nagashima F. Chemical constituents from two New Zealand liverworts, Paraschistochila pinnatifolia and of bryophytes: Bio- and chemical diversity, biological activity and Trichocolea mollissima. J Nat Prod. 1997; 60: 421–424. chemosystematics. In: Kinghorn DA, Falk H, Kobayashi J. eds. Progress 31. Perry NB, Burgess E J, Tangney RS. Cytotoxic 8,9-secokaurane in the chemistry of organic natural products. Springer, Vienna, 2013.p. diterpenes from a New Zealand Liverwort, Lepidolaena taylorii. 1–796. Tetrahedron Lett. 1996; 37: 9387–9390. 5. Ludwiczuk A, Asakawa Y. Distribution of terpenoids and aromatic 32. Perry NB, Burgess EJ, Baek SH, Weavers RT, Geis W, Mauger AB. compounds in selected southern hemispheric liverworts. Fieldiana 1999. 11-Oxygenated cytotoxic 8,9-secokauranes from a New Zealand Bot. 2008; 47: 37–58. liverwort, Lepidolaena taylorii. Phytochemistry 1999; 50: 423–433. 6. Ludwiczuk A, Asakawa Y. Chemosystematics of the liverworts collected 33. Nagashima F., Kondoh M, Uematsu T, Nishiyama A, Saito S, Sato M, in Borneo. Tropic Bryol. 2010; 31: 33–42. et al. Cytotoxic and apoptosis-Inducing ent-kaurane-type diterpenoids 7. Ludwiczuk A, Nagashima F, Gradstein SR, Asakawa Y. Volatile from the Japanese liverwort Jungermannia trunctata Nees. Chem Pharm Components from the selected Mexican, Ecuadorian, Greek, German Bull. 2002; 50: 808–813. and Japanese liverworts. Nat Prod Commun. 2008; 3: 133–140. 34. Perry NB, Burgess EJ, Foster LM, Gerard PJ, Toyota M, Asakawa Y., 8. Ludwiczuk A, Komala I, Pham A, Bianchini JP, Raharivelomanana P, 2008. Insect antifeedant sesquiterpene acetals from the liverwort Asakawa Y. Volatile components from selected Tahitian liverworts. Lepidolaena clavigera. 2. Structures, artifacts and activity. J Nat Prod. Nat. Prod. Commun. 2009; 4: 1387–1392. 1998; 71: 258–261. 9. Asakawa Y. Phytochemistry of bryophytes: biologically active 35. Perry NB, Burgess EJ, Baek SH, Weavers RT. The First atisane terpenoids and aromatic compounds from liverworts. In: Romeo J. diterpenoids from a liverwort: Polyols from Lepidolaena clavigera. ed. Phytochemicals in human health protection, nutrition, and plant Org Lett. 2001; 3: 4243–4245. defense. New York, Kluwer Academic/Plenum Publishers, 1999.p.319– 36. Neves M, Morais R, Gafner S, Hostettmann K. Three triterpenoids and 342. one flavonoid from the liverwort Asterella blumeana grown in vitro. 10. Asakawa Y. Liverworts – potential source of medicinal compounds. Phytotherapy Res. 1998; 12: 21–24. Curr Pharm Design. 2008; 14: 3067–3088. 37. Wong SM, Oshima Y, Pezzuto J, Fong H, Farnsworth N. Plant anticancer 11. Asakawa Y. Recent advances of biologically active substances from agents XXXIX. Triterpenes from Isis missouriensis (Iridaceae). J Pharm Marchantiophyta. Nat Prod Commun. 2008; 3: 77–92. Sci. 1986; 75: 317–320. 12. Asakawa Y. Biologically active substances from bryophytes. In: 38. Komala I, Ito T, Nagashima F, Yagi Y, Asakawa Y. Cytotoxic bibenzyls, Chopra RN, Bhatla SC. eds. development: Physiology and germacrane- and pinguisane-type sesquiterpenoids from the biochemistry. Boca Raton, CRC Press, 1990.p.259–287. Indonesian, Tahitian and Japanese liverworts. Nat Prod Commun. 13. Asakawa Y. Terpenoids and aromatic compounds with pharmacological 2011; 6: 303–309. activity from bryophytes. In: Zinsmeister DH, Mues R, eds. Bryophytes: 39. Asakawa Y, Toyota M, Tori M, Hashimoto T. Chemical structures Their chemistry and chemical taxonomy. Oxford, Oxford University of macrocyclic bis(bibenzyls) isolated from liverworts (Hepaticae). Press, 1990.p.369–410. Spectroscopy 2000; 14: 149–175. Journal of Pre-Clinical and Clinical Research, 2013, Vol 7, No 2 85 Yoshinori Asakawa, Agnieszka Ludwiczuk, Toshihiro Hashimoto. Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

40. Asakawa Y, Matsuda R. Riccardin C, a novel cyclic bibenzyl derivative 56. Kondoh M, Suzuki I, Harada M, Nagashima F, Fuji M, Asakawa Y. from Reboulia hemisphaerica. Phytochemistry 1982; 21: 2143–2144. et al. Activation of p38 mitogen-activated protein kinase during ent- 41. Huang WJ, Wu CL, Lin CW, Chi LL, Chen PY, Chiu CJ, et al. Marchantin 11α-hydroxy-16-kauren-15-one-induced apoptosis in human leukemia A, a cyclic bis(bibenzyl ether), isolated from the liverwort Marchantia HL-60 cells. Planta Med. 2005; 71: 275–277. emarginata subsp. tosana induces apoptosis in human MCF-7 breast 57. Izumi S, Nishio Y, Takashima O, Hirata T. Monoterpenoids, potent cancer cells. Cancer Lett. 2010; 291: 108–119. inducers of apoptosis in the cells of Marchantia polymorpha. Chem 42. Xie CF, Qu JB, Wu XZ, Liu N, Ji M, Lou HX. Antifungal macrocyclic Lett. 1997; 837–838. bis(bibenzyls) from the Chinese liverwort intermedium 58. Guo DX, Du Y, Wang YY, Sun LM, Qu JB, Wang XN, et al. Secondary L. Nat Prod. Res. 2010; 24; 515–520. metabolites from the liverwort Ptilidium pulcherrimum. Nat Prod 43. Shi YQ, Liao YX, Qu XJ, Yuan HQ, Li S, Qu JB, et al. Marchantin C, a Commun. 2009; 4: 1319–1322. macrocyclic bisbibenzyl, induces apoptosis of human glioma A172 cells. 59. Nozaki H, Hayashi KI, Nishimura N, Kawaide H, Matsuo A, Takaoka Cancer Lett. 2008; 262: 173–182. D. 2007. Momilactone A and B as allelochemicals from moss Hypnum 44. Shi YQ, Qu XJ, Liao YX, Xie CF, Cheng YN, Li S. et al. Reversal effect plumaeforme: First occurrence in bryophytes. Biosci Biotechnol of a macrocyclic bisbibenzyl plagiochin E on multidrug resistance in Biochem 2007; 71: 3127–3130. adriamycin-resistant K562/A02 cells. Eur. J. Pharmacol. 2008; 584: 60. Kim SJ, Park HR, Park E, Lee SC. Cytotoxic and antitumor activity of 66–71. momilactone B from rice hulls. J Agric Food Chem. 2007; 55: 1702–1706. 45. Speicher A, Groh M, Zapp J, Schaumloffel A, Knauer M, Bringmann G.. 61. Zheng G.Q, Ho DK, Elder PJ, Stephens RE, Cottrell CE, Cassady JM. Synthesis-driven structure revision of ‘plagiochin E’, a highly bioactive Ohioensins and pallidisetins: Novel cytotoxic agents from the moss bisbibenzyl. Synlett. 2009; 1852–1858. Polytrichum pallidisetum. J Nat Prod. 1994; 57: 32–41. 46. Lorimer SD, Perry NB. Antifungal hydroxyacetophenones from the 62. Fu P, Lin S, Shan L, Shen, YH, Tang J, Liu RH, et al. Constituents of the New Zealand liverwort, Plagiochila fasciculata. Planta Med. 1994; 60: moss Polytrichum commune. J Nat Prod. 2009; 72: 1335–1337. 386–387. 63. Asakawa Y, Ludwiczuk, A. Distribution of cyclic bis-bibenzyls in 47. Perry NB, Foster LM, Lorimer SD, May BCH, Weavers RT, Toyota M, the Marchantiophyta (liverworts), and higher plants and their et al. Isoprenyl phenyl ethers from liverworts of the Trichocolea: biological activities, biosynthesis, and total synthesis. Heterocycles. Cytotoxic activity, structural corrections, and synthesis. J Nat Prod. 2012; 86: 891–917. 1996; 59: 729–733. 64. Iwai Y, Murakami K, Gomi Y, Hashimoto T, Asakawa Y, Okuno Y, 48. Baek SH, Oh HJ, Lim JA, Chun HJ, Lee HO, Ahn JW, et al. Biological et al. Anti-influenza activity of marchantins, macrocyclic bisbibenzyls activities of Methyl-4-[[(2E)-3,7-dimethyl-2,6-octadienyl]oxy]-3- contained in liverworts. PLoS ONE. 2011; 6: 19825. hydroxybenzoate. Bull. Korean Chem Soc. 2004; 25: 195–197. 65. Yoshida T, Hashimoto T, Takaoka S, Kan Y, Tori M, Asakawa Y. Phenolic 49. Nagashima F, Kasai W, Kondoh M, Fujii M. Watanabe Y, Braggins J. E, constituents of the liverwort: Four novel cyclic Bisbibenzyl dimers from et al. New ent-kaurene-type diterpenoids possessing cytotoxicity from Blasia pusilla L. Tetrahedron 1996; 52: 14487–14500. the New Zealand liverwort Jungermannia species. Chem Pharm Bull. 66. Shi YQ, Zhu CJ, Yuan HQ, Li BQ, Gao J, Qu XJ, et al. Marchantin C, 2003; 51: 1189–1192. a novel microtubule inhibitor from liverwort with anti-tumor activity 50. Nagashima F, Kondoh M, Fujii M, Takaoka S, Watanabe Y, Asakawa both in vivo and in vitro. Cancer Lett. 2009; 276: 160–170. Y. Novel cytotoxic diterpenoids from the New Zealand liverwort 67. Morita H, Tomizawa Y, Tsuchiya T, Hirasawa Y, Hashimoto T, Asakawa Jungermannia species. Tetrahedron 2005; 51: 4531–4544. Y. Antimitotic activity of two macrocyclic bis(bibenzyls), isoplagiochins 51. Nagashima F, Kondoh M, Kawase M, Simizu S, Osada H, Fuji M, et al. A and B from the liverwort Plagiochila fruticosa. Bioorg Med Chem Apoptosis-inducing properties of ent-kaurene-type diterpenoids from Lett. 2009; 19: 493–496. the liverwort Jungermannia trunctata. Planta Med. 2003; 69: 377–379. 68. Hashimoto T, Yasuda A, Akazawa K, Takaoka S, Tori M, Asakawa Y. 52. Kondoh M, Suzuki I, Sato M, Nagashima F, Simizu S, Harada M.et al. Three novel dimethyl pyrroledicarboxylate, lycogarubin A-C, from 2004. Kaurene diterpene induces apoptosis in human leukemia cells the Myxomycetes Lycogala epidendrum. Tetrahedron Lett. 1994; 35: partly through a caspase-8-dependent pathway. J Pharmacol Exp Ther. 2559–2560. 2004; 311: 115–122. 69. Quang DN, Hashimoto T, Asakawa Y. Inedible mushrooms, a good 53. Suzuki I, Kondoh M, Harada M, Koizumi N, Fujii M, Nagashima F, source of biologically active compounds. J Chem Rec. 2006; 6: 79–99. et al. An ent-kaurene diterpene enhances apoptosis induced by tumor 70. Asakawa Y, Hashimoto T, Mizuno Y, Tori M. Cryptoporic acids A-G, necrosis factor in human leukemia cells. Planta Med. 2004; 70: 723–727. drimane-type sesquiterpenoid ethers of isocitoric acid from the fungus 54. Suzuki I, Kondoh M, Nagashima F, Fujii M, Asakawa Y, Watanabe Y. Cryptoporus volvatus. Phytochemistry 1992; 31: 579–592. A comparison of apoptosis and necrosis induced by ent-kaurene-type 71. Buchanan MS, Hashimoto T, Asakawa Y. Cytochalasins from a Daldinia diterpenoids in HL-60 cells. Planta Med. 2004; 70: 401–406. sp. of fungus. Phytochemistry 1996; 41: 821–828. 55. Kondoh M, Nagashima F, Suzuki I, Harada M, Fuji M, Asakawa Y, et al. 72. Kashiwada Y, Yamazaki K, Ikeshiro Y, Yamagishi T, Fujioka T, Mihashi Induction of apoptosis by new ent-kaurene-type diterpenoids isolated K, et al. 2001. Isolation of rhododaurichromanic acid B and the anti- from the New Zealand liverwort Jungermannia species. Planta Med. HIV principles rhododaurichromanic acid A and rhododaurichromenic 2005; 71: 1005–1009. acid from Rhododendron dauricum. Tetrahedron. 2001; 57:1559–15.